Testosterone is the major androgenic hormone. It is responsible for the development of the male external genitalia and secondary sexual characteristics. In females, its main role is as an estrogen precursor. In both genders, it also exerts anabolic effects and influences behavior.
In men, testosterone is secreted by the testicular Leydig cells and, to a minor extent, by the adrenal cortex. In premenopausal women, the ovaries are the main source of testosterone with minor contributions by the adrenals and peripheral tissues. After menopause, ovarian testosterone production is significantly diminished. Testosterone production in testes and ovaries is regulated via pituitary-gonadal feedback involving luteinizing hormone (LH) and, to a lesser degree, inhibins and activins.
Most circulating testosterone is bound to sex hormone-binding globulin (SHBG), which in men also is called testosterone-binding globulin. A lesser fraction is albumin bound and a small proportion exists as free hormone. Historically, only the free testosterone was thought to be the biologically active component. However, testosterone is weakly bound to serum albumin and dissociates freely in the capillary bed, thereby becoming readily available for tissue uptake. All non-SHBG-bound testosterone is therefore considered bioavailable.
During childhood, excessive production of testosterone induces premature puberty in boys and masculinization in girls. In adult women, excess testosterone production results in varying degrees of virilization, including hirsutism, acne, oligo-amenorrhea, or infertility. Mild-to-moderate testosterone elevations are usually asymptomatic in males, but can cause distressing symptoms in females. The exact causes for mild-to-moderate elevations in testosterone often remain obscure. Common causes of pronounced elevations of testosterone include genetic conditions (eg, congenital adrenal hyperplasia); adrenal, testicular, and ovarian tumors; and abuse of testosterone or gonadotrophins by athletes.
Decreased testosterone in females causes subtle symptoms. These may include some decline in libido and nonspecific mood changes. In males, it results in partial or complete degrees of hypogonadism. This is characterized by changes in male secondary sexual characteristics and reproductive function. The cause is either primary or secondary/tertiary (pituitary/hypothalamic) testicular failure. In adult men, there also is a gradual modest, but progressive, decline in testosterone production starting between the 4th and 6th decades of life. Since this is associated with a simultaneous increase of SHBG levels, bioavailable testosterone may decline more significantly than apparent total testosterone, causing nonspecific symptoms similar to those observed in testosterone deficient females. However, severe hypogonadism, consequent to aging alone, is rare.
Current methods for measuring free testosterone in biological fluid such as blood rely on measuring total testosterone, then measuring the amount of Sex Hormone Binding Globulin (SHBG) which binds the testosterone which is not free, and in some cases also measuring albumin (since testosterone can be bound by this as well), and then calculating an approximate amount of free testosterone by subtracting the amount theoretically bound to SHBG and the amount theoretically bound to albumin from the total measured testosterone. SHBG binds other sex hormones besides testosterone, so the assumption that the amount of SHBG equals the amount of bound testosterone is not accurate. This is also true for albumin which binds thousands of ligands non-specifically. This process is laborious, inaccurate, not reproducible with repeat measurements (high CVs), and requires the extra steps of measuring SHBG levels and albumin solely for the purpose of estimating how much of the total testosterone is free, which is presumed to be bio-available or bio-active. However, even if this method were to accurately measure the free testosterone, which it doesn't, free testosterone is not equivalent to bio-active testosterone when various cell reporter assays for bio-activity are used. Cell-based reporter assays are not suitable for clinical diagnostic use although they are useful as validation testing to demonstrate that a given assay such as described herein accurately measures bioavailable bioactive testosterone.
Measurement of total testosterone is often sufficient for diagnosis of morbidities or abnormalities, particularly if it is combined with measurements of LH and follicle-stimulating hormone (FSH). However, these tests are insufficient for diagnosis of mild abnormalities of testosterone homeostasis, particularly if abnormalities in SHBG function or levels are present. Therefore, additional methods for the measurements of free testosterone or bioavailable testosterone are needed in the art.